Nature Biotechnology
19, 137 - 141 (2001)
doi:10.1038/84397
A high signal-to-noise Ca2+ probe composed of a single green fluorescent proteinJunichi Nakai, Masamichi Ohkura
& Keiji Imoto
Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, 444-8585, Japan.
Correspondence should be addressed to Junichi Nakai jnakai@nips.ac.jpcalciumgreen fluorescent proteincalmodulinmyosin light chain kinaseprotein−protein interactionphotoisomerizationRecently, several groups have developed green fluorescent protein (GFP)-based Ca2+ probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca2+ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K
d for Ca2+ of 235 nM. Association kinetics of Ca2+ binding were faster at higher Ca2+ concentrations, with time constants decreasing from 230 ms at 0.2  M Ca2+ to 2.5 ms at 1 M Ca2+. Dissociation kinetics (  200 ms) are independent of Ca2+ concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
|
